AGL86 Antibody

What is AGL86 and why are antibodies against it important for plant research?

AGL86 is a MADS-box transcription factor encoded by the AT1G31630 gene in Arabidopsis thaliana. It has been identified in screens for transcription factors that provide tolerance to 9-HOT, a plant oxylipin involved in stress responses . The protein belongs to a large family of plant-specific transcription factors that regulate various developmental processes and stress responses.

Antibodies against AGL86 are critical research tools for several reasons. First, they enable visualization and quantification of endogenous AGL86 protein levels across different tissues and experimental conditions. Second, they facilitate chromatin immunoprecipitation (ChIP) experiments to identify genomic binding sites of AGL86. Third, they allow isolation of protein complexes through co-immunoprecipitation to identify interaction partners. These applications are essential for understanding AGL86's role in transcriptional networks regulating plant stress responses, particularly in oxylipin signaling pathways.

What are the available types of antibodies for AGL86 detection?

For MADS-box transcription factors like AGL86, researchers typically have access to several antibody types:

• Polyclonal antibodies: Generated by immunizing animals (typically rabbits) with purified AGL86 protein or synthetic peptides corresponding to unique regions of AGL86 . These recognize multiple epitopes, providing robust detection but potentially lower specificity.

• Monoclonal antibodies: Produced from a single B-cell clone, these recognize a single epitope on AGL86, offering higher specificity but potentially lower sensitivity for low-abundance transcription factors.

• Recombinant antibodies: Engineered antibodies with defined binding properties, including single-chain variable fragments (scFvs) or antigen-binding fragments (Fabs).

The choice depends on the specific application, with polyclonal antibodies often preferred for initial detection and monoclonal or recombinant antibodies for applications requiring higher specificity.

How is the specificity of AGL86 antibodies validated?

Proper validation of AGL86 antibodies is crucial for reliable experimental results. A comprehensive validation approach includes:

• Western blot analysis comparing wild-type plants with AGL86 knockout or knockdown mutants to confirm specificity.

• Immunoprecipitation followed by mass spectrometry to confirm that the antibody captures the intended protein.

• Preabsorption tests with the immunizing antigen to demonstrate that binding is epitope-specific.

• Cross-reactivity testing with related MADS-box proteins, particularly those with high sequence homology.

• Orthogonal validation using multiple antibodies targeting different epitopes of AGL86.

• Correlation of protein detection with known mRNA expression patterns from transcriptomic data.

Each validation method addresses different aspects of antibody specificity, and combining multiple approaches provides the strongest evidence for antibody reliability in AGL86 research applications.

What are the optimal sample preparation methods for detecting AGL86 in plant tissues?

For effective detection of MADS-box transcription factors like AGL86 in plant tissues, specialized extraction protocols are essential:

• Nuclear extraction is recommended since AGL86 is predominantly nuclear-localized:

  • Grind tissue in liquid nitrogen

  • Extract using nuclear isolation buffers (e.g., 50mM HEPES pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 10% glycerol)

  • Include protease inhibitor cocktails to prevent degradation

  • Centrifuge to isolate nuclear fraction

  • Solubilize nuclear proteins with appropriate detergents

• Tissue selection considerations:

  • Young, actively growing tissues typically show higher transcription factor expression

  • Consider stress conditions that might induce AGL86 expression based on its role in oxylipin response

  • Compare tissues with different known expression levels as internal controls

• Fixation for immunohistochemistry:

  • 4% paraformaldehyde fixation preserves protein localization while maintaining epitope accessibility

  • Antigen retrieval may be necessary if fixation reduces antibody binding

Proper sample preparation significantly impacts the success of downstream applications such as western blotting, immunoprecipitation, and chromatin immunoprecipitation with AGL86 antibodies.

How can AGL86 antibodies be used in ChIP assays to identify binding targets?

Chromatin immunoprecipitation (ChIP) using AGL86 antibodies allows identification of genomic regions bound by this transcription factor. A methodological workflow includes:

• Cross-linking: Treat plant tissue with formaldehyde (typically 1%) to stabilize protein-DNA interactions.

• Chromatin extraction and fragmentation:

  • Extract chromatin from nuclei

  • Sonicate or enzymatically digest to generate 200-500bp fragments

  • Verify fragmentation by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate with validated AGL86 antibodies (typically 2-5μg per reaction)

  • Include appropriate controls (IgG, input samples, no-antibody)

  • Capture antibody-chromatin complexes with protein A/G beads

• Washing and elution:

  • Use increasingly stringent washes to reduce background

  • Elute protein-DNA complexes and reverse cross-links

  • Purify DNA for downstream analysis

• Analysis options:

  • ChIP-qPCR for known or candidate target genes

  • ChIP-seq for genome-wide identification of binding sites

  • Bioinformatic analysis to identify binding motifs and associated genes

For plant MADS-box factors like AGL86, optimizing sonication conditions and antibody concentrations is particularly important due to their often moderate expression levels and the complex plant chromatin environment .

What dilutions and conditions are optimal for immunoblot detection of AGL86?

For optimal immunoblot detection of MADS-box transcription factors like AGL86, careful optimization of experimental conditions is necessary:

Additional considerations:

• Sample loading: Load 30-50μg of nuclear protein extract per lane for optimal detection.

• Transfer conditions: Wet transfer at 30V overnight often yields better results for transcription factors than rapid transfer protocols.

• Membrane selection: PVDF membranes (0.45μm) typically provide better sensitivity than nitrocellulose for low-abundance transcription factors.

• Signal enhancement: Consider using signal enhancement systems (e.g., biotin-streptavidin amplification) for detecting low-abundance AGL86, particularly in tissues with limited expression.

Systematic optimization of these parameters should be performed when establishing a new immunoblot protocol for AGL86 detection.

How can AGL86 antibodies be used to investigate protein-protein interactions within MADS-box transcription factor networks?

MADS-box transcription factors typically function in combinatorial protein complexes. AGL86 antibodies enable several approaches to investigate these interactions:

• Co-immunoprecipitation (Co-IP):

  • Prepare native protein extracts under non-denaturing conditions

  • Immunoprecipitate with AGL86 antibodies

  • Analyze co-precipitated proteins by western blot (for known interactions) or mass spectrometry (for unbiased discovery)

  • Include appropriate controls (IgG, lysates from knockout plants)

• Sequential ChIP (re-ChIP):

  • Perform first ChIP with AGL86 antibodies

  • Elute complexes under non-denaturing conditions

  • Perform second ChIP with antibodies against suspected interaction partners

  • Analyze enriched DNA to identify co-occupied genomic regions

  • This approach reveals functional interactions at chromatin

• Proximity ligation assay (PLA):

  • Use primary antibodies against AGL86 and potential interaction partners

  • Apply oligonucleotide-conjugated secondary antibodies

  • Amplify signal when proteins are in close proximity (<40nm)

  • Visualize interaction sites by fluorescence microscopy

  • Quantify interaction frequency in different cell types or conditions

These complementary approaches can reveal both stable and transient interactions of AGL86 with other transcriptional regulators, particularly in the context of stress responses where it was identified as a factor involved in oxylipin tolerance .

What protocols exist for using AGL86 antibodies in plant immunohistochemistry applications?

Immunohistochemical detection of AGL86 in plant tissues requires specialized protocols:

• Tissue fixation and embedding:

  • Fix tissues in 4% paraformaldehyde in PBS (pH 7.4) for 4-6 hours

  • Dehydrate through an ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

  • Embed in paraffin or LR White resin for sectioning

  • Cut 5-10μm sections and mount on adhesive slides

• Antigen retrieval:

  • For paraffin sections, deparaffinize with xylene and rehydrate

  • Perform heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • For plant tissues, additional cell wall digestion with pectinase/cellulase may improve antibody accessibility

• Immunostaining procedure:

  • Block with 5% normal serum and 0.3% Triton X-100 in PBS (2 hours)

  • Incubate with primary AGL86 antibody (1:100 to 1:500 dilution) overnight at 4°C

  • Wash thoroughly (3-5 times, 10 minutes each) with PBS containing 0.1% Tween-20

  • Apply fluorophore-conjugated secondary antibodies (1:200 to 1:500) for 2 hours

  • Counterstain nuclei with DAPI (1μg/ml)

  • Mount in anti-fade medium

• Controls and validation:

  • Include negative controls (primary antibody omission, non-immune IgG)

  • Use tissues from AGL86 knockout plants as specificity controls

  • Compare staining patterns with in situ hybridization data for AGL86 mRNA

These protocols can be adapted for confocal microscopy, allowing co-localization studies with other nuclear proteins or chromatin markers to investigate AGL86 function in specific cell types during plant development or stress responses.

How can single-cell approaches with AGL86 antibodies reveal heterogeneity in transcription factor dynamics?

Advanced single-cell methodologies using AGL86 antibodies can uncover cell-type-specific regulation:

• Single-cell immunofluorescence analysis:

  • Perform high-resolution imaging of plant tissues using AGL86 antibodies

  • Quantify nuclear signal intensity across different cell types

  • Correlate with cell-specific markers to identify patterns of expression

  • Track changes in subcellular localization in response to environmental stimuli

• CUT&Tag or CUT&RUN with AGL86 antibodies:

  • These techniques use antibody-directed nuclease activity to map protein binding sites

  • Require fewer cells than conventional ChIP (can be adapted for specific cell types)

  • Provide higher resolution data on genomic binding locations

  • Can reveal cell-type-specific target genes of AGL86

• Integration with single-cell transcriptomics:

  • Isolate nuclei from specific cell types using fluorescence-activated nucleus sorting

  • Perform parallel analyses of AGL86 binding (CUT&Tag) and gene expression (RNA-seq)

  • Correlate binding patterns with transcriptional output

  • Identify cell-type-specific regulatory networks

These approaches are particularly valuable for understanding AGL86 function in complex plant tissues, where its activity may vary between different cell types during development or in response to stresses like oxylipin exposure that have been shown to involve AGL86 .

How can non-specific binding be reduced when using AGL86 antibodies?

Non-specific binding is a common challenge with transcription factor antibodies, including those targeting AGL86:

• Causes of non-specific binding:

  • Cross-reactivity with related MADS-box proteins (high sequence homology in DNA-binding domains)

  • Interaction with abundant proteins in plant extracts

  • Suboptimal blocking or washing conditions

• Optimization strategies:

  • Pre-adsorb antibodies with plant extracts from AGL86 knockout plants

  • Increase blocking stringency (longer time, different blocking agents)

  • Add competing proteins (e.g., BSA) to antibody dilution buffer

  • Optimize salt concentration in wash buffers (150-500mM NaCl)

  • Include non-ionic detergents (0.1-0.3% Triton X-100) in wash buffers

• Troubleshooting specific problems:

Systematic optimization of these parameters can significantly improve signal-to-noise ratio when working with AGL86 antibodies, particularly in complex plant extracts where many related MADS-box proteins may be present .

What controls are essential when using AGL86 antibodies in experimental procedures?

Proper controls are critical for interpreting results obtained with AGL86 antibodies:

• Essential negative controls:

  • Samples from AGL86 knockout or knockdown plants

  • Non-immune IgG from the same species as the primary antibody

  • Primary antibody omission controls

  • Peptide competition assays (pre-incubation with immunizing antigen)

• Positive controls:

  • Recombinant AGL86 protein (for western blots)

  • Tissues known to express AGL86 based on transcriptomic data

  • Epitope-tagged AGL86 expressed in transgenic plants

• Application-specific controls:

  • For ChIP: Input samples, IgG controls, positive control regions (known targets)

  • For immunohistochemistry: Tissues with known expression patterns

  • For co-immunoprecipitation: Stringency controls with different wash conditions

• Validation across methods:

  • Correlate protein detection with mRNA expression data

  • Confirm key findings with orthogonal detection methods

  • Use multiple antibodies targeting different epitopes when possible

Implementing these controls systematically ensures reliable interpretation of results obtained with AGL86 antibodies, particularly important when studying members of large transcription factor families like MADS-box proteins in plants .

How can researchers address batch-to-batch variability in AGL86 antibodies?

Batch-to-batch variability can significantly impact experimental reproducibility when using antibodies against transcription factors like AGL86:

• Characterization of new antibody batches:

  • Perform side-by-side western blots with previous batches

  • Titrate each new batch to determine optimal working dilutions

  • Create standard curves using recombinant AGL86 protein

  • Document detection limits and dynamic range

• Reference standards and normalization:

  • Maintain aliquots of reference samples (e.g., nuclear extracts from specific tissues)

  • Create internal calibration standards with recombinant protein

  • Normalize signals to housekeeping proteins or total protein stains

  • Consider absolute quantification using spike-in controls

• Long-term mitigation strategies:

  • Purchase larger lots of validated antibodies when available

  • Aliquot antibodies to minimize freeze-thaw cycles

  • Document lot numbers and validation data for each experiment

  • Consider developing monoclonal antibodies for critical applications

• Data reporting practices:

  • Report antibody source, catalog number, and lot number in publications

  • Include validation data in supplementary materials

  • Describe specific optimization steps required for each batch

  • Be transparent about limitations of the antibodies used

These approaches help maintain experimental consistency despite the inherent variability in antibody production, particularly important for quantitative applications of AGL86 antibodies in plant research.

How are AGL86 antibodies being used to investigate plant stress response mechanisms?

AGL86 was identified in screens for transcription factors providing tolerance to 9-HOT, implicating it in plant stress responses . Antibodies are enabling several research directions in this area:

• Stress-responsive dynamics:

  • Tracking AGL86 protein accumulation, degradation, and post-translational modifications during stress exposure

  • Comparing AGL86 protein levels across different stress conditions (drought, heat, pathogen attack)

  • Correlating AGL86 levels with stress tolerance phenotypes

  • Examining tissue-specific stress responses mediated by AGL86

• Stress-specific target gene identification:

  • ChIP-seq with AGL86 antibodies under normal versus stress conditions

  • Identifying condition-specific binding events that may explain stress tolerance

  • Integrating binding data with stress-responsive transcriptomics

  • Validating direct regulation of stress-responsive genes

• Regulatory network mapping:

  • Co-immunoprecipitation under stress conditions to identify stress-specific protein interactions

  • Re-ChIP experiments to map combinatorial binding with other stress-responsive transcription factors

  • Reconstruction of regulatory cascades involving AGL86 during stress responses

These applications provide mechanistic insights into how AGL86 contributes to stress tolerance, particularly in oxylipin response pathways where it was originally identified as a regulatory factor .

What are the technical challenges in developing phospho-specific antibodies for AGL86?

MADS-box transcription factors including AGL86 are often regulated by phosphorylation. Developing phospho-specific antibodies presents several challenges:

• Identification of physiologically relevant phosphorylation sites:

  • Mass spectrometry analysis of AGL86 under different conditions

  • Bioinformatic prediction based on conserved kinase motifs

  • Comparison with known regulatory phosphorylation sites in related MADS-box proteins

  • Functional validation of candidate sites through mutagenesis

• Antibody design considerations:

  • Selection of peptide sequences surrounding the phosphorylation site

  • Ensuring specificity for the phosphorylated versus non-phosphorylated state

  • Addressing potential cross-reactivity with similar phosphorylation sites in related proteins

  • Balancing epitope length to maintain specificity while ensuring accessibility

• Validation strategies:

  • Testing against phosphorylated and dephosphorylated (phosphatase-treated) samples

  • Comparing reactivity with phosphomimetic (S/T→D/E) and phospho-null (S/T→A) mutants

  • Verification of signal changes in response to kinase or phosphatase inhibitors

  • Correlation with known biological stimuli that trigger phosphorylation

• Application-specific considerations:

  • Optimizing sample preparation to preserve phosphorylation status

  • Including phosphatase inhibitors in extraction buffers

  • Adapting immunoprecipitation protocols for phosphorylated proteins

  • Developing quantitative assays for phosphorylation dynamics

These phospho-specific antibodies would enable researchers to track the activation state of AGL86 during development and stress responses, providing deeper insights into its regulatory mechanisms.

How might AGL86 antibodies contribute to understanding evolutionary conservation of MADS-box factor function across plant species?

AGL86 antibodies can serve as valuable tools for comparative studies across plant species:

• Cross-species reactivity analysis:

  • Testing antibody recognition of AGL86 orthologs in different plant species

  • Mapping epitope conservation across evolutionary distance

  • Establishing detection protocols for non-model species

  • Creating conservation maps of recognizable domains

• Functional conservation studies:

  • Comparing subcellular localization patterns across species

  • Identifying conserved protein interaction partners through co-immunoprecipitation

  • Mapping conserved versus species-specific genomic binding sites

  • Correlating binding site conservation with regulatory outcomes

• Stress response comparison:

  • Examining AGL86-like protein expression in stress-tolerant versus susceptible species

  • Tracking evolutionary adaptations in post-translational modification patterns

  • Identifying lineage-specific innovations in regulatory networks

  • Correlating molecular differences with physiological adaptations

• Methodological approaches:

  • Develop degenerate epitope antibodies that recognize conserved domains

  • Establish systematic validation pipelines for cross-species applications

  • Create databases of cross-reactivity patterns and epitope conservation

  • Design peptide arrays to map recognition profiles across species

These evolutionary approaches can reveal fundamental principles of MADS-box factor function while also identifying species-specific adaptations, particularly in stress response pathways where AGL86 has been implicated .